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SEVENTH FRAMEWORK PROGRAMME


Workpackage 08

Workpackage Objectives

  • To determine the effects of bumetanide and other GABA acting drugs -including phenobarbital and midazolam on the generation, propagation and effects of seizures using the triple chamber and interconnected hippocampi in vitro. We shall determine if these drugs alone or combined with bumetanide alter the formation of an epileptogenic mirror focus during seizures. These studies will provide a rationale for the comparison of the anti- epileptic actions of bumetanide with and without GABA. We shall specifically compare the actions of bumetanide /Phenobarbital after a few or more seizures to determine whether their effects depend on seizure induced alterations of intracellular chloride and hence shifts of GABA to excitation.
  • To determine the acute effects of bumetanide on the physiological patterns of activity in the immature cortex, using in vivo recordings from the neocortex and hippocampus. We shall centre this study on GABA signalling and the excitatory effects of GABA that have so far been evidenced only by in vitro systems. We shall therefore provide direct measures of the actions of bumetanide on GABA in vivo.

Workpackage Lead

Academic Lead:  Prof. Yehezkel Ben-Ari (INSERM/INMED)

Workpackage description 

The main goal of this work package is to determine whether bumetanide exerts adverse events on the physiological patterns of activity in the naïve and epileptic immature cortex.  A wide range of techniques and preparations, many of which have been developed by INMED (INSERM) researchers will be used including the triple chamber with the two intact hippocampi, multiple field potential and multiple unit recordings, single channel recordings and biphoton calcium or chloride imaging from neocortical slices. These results will be presented to the co-sponsor (WP 11) and will enhance the planned PUMA application. We shall concentrate on in vitro experiments as they condition our understanding of the actions of bumetanide and this is much needed in order to implement its use. Specifically, we shall examine in detail whether the antiepileptic actions of bumetanide are conditioned by the number of seizures that are generated prior to its usage. 

Task 1: Effect of bumetanide on epileptogenesis in the mirror-focus model.

We will induce secondary epileptic mirror-focus in the contralateral hippocampus using interconnected hippocampi preparations obtained from the neonatal rats (age groups P6-7), by repetitive application of kainate to the ipsilateral hippocampus. The contralateral propagation of seizures and the success rate of mirror-focus induction will be compared between the control conditions and in the presence of bumetanide in the contralateral chamber during the mirror-focus induction. This experiment will enable us to determine whether bumetanide exerts antiepileptogenic action.

Task 2: Anticonvulsive effect of bumetanide in the mirror-focus model.

Following secondary epileptic mirror-focus induction in the control conditions (as described in task 2), we will determine the effect of bumetanide on the epileptiform activity in the contralateral hippocampus. In the same experiment, we will also determine the effect of bumetanide on Egaba in the mirror focus neurons using methodology described in task 1. We will also determine the anticonvulsive efficacy of Phenobarbital and midazolam in the mirror focus model and combined treatments with bumetanide. Paired recordings from the two interconnected hippocampi will enable us to determine the effects of bumetanide on the generation of seizures, their propagation and their effects determined by the formation of an epileptogenic mirror focus. Of particular importance will be the determination of the effects of bumetanide and phenobarbitone or midazolam on high frequency oscillations that are required for seizures to propagate.

WP08 - Preclinical studies